Extended Data Fig. 6: SEC14L2 promotes endosomal PI4P removal in parallel with VAPA/B-OSBP machinery.
From: A Golgi-derived vesicle potentiates PtdIns4P to PtdIns3P conversion for endosome fission
a-d, GI-SIM imaging of SEC14L2 KO COS-7 cells overexpressing iRFP-FRB-RAB5 (green and gray) and mEmerald-KDEL (magenta) at 1-s intervals after endosomal recruitment of VPS34 or SAC1ΔTMD. The main panel shows a partial cell periphery with endosome fission. The insets illustrate a single fission event. White arrows mark approximate sites of fission. Time 0 corresponds to the frame in which tubules first emerge. e, Summary of ER-endosome contact duration before endosome fission in (a-d). SEC14L2 KO COS-7 cells were transfected with mCherry-FKBP, mCherry-FKBP-VPS34, or mCherry-FKBP-SAC1ΔTMD in combination with iRFP-FRB-RAB5 and mEmerald-KDEL. After 1 μM rapamycin (Rap.) treatment for 30 min, cells were examined for endosome fission. n = 20 fission events from 8 WT cells with mCherry-FKBP transfection, n = 26 fission events from 13 SEC14L2 KO cells with mCherry-FKBP transfection, n = 22 fission events from 6 SEC14L2 KO cells with mCherry-FKBP-VPS34 transfection, and n = 48 fission events from 22 SEC14L2 KO mCherry-FKBP-SAC1ΔTMD transfection. Data are presented as the mean ± SD. Unpaired, Two-tailed student’s t-test was used. ***p = 6.47E-07. f, Confocal images of GFP-PHOSBP in WT and VAPA + VAPB siRNA or OSBP siRNA transfected COS-7 cells with or without zSec14l3-mCherry overexpression. Statistics are shown in Fig. 6c. g, Confocal images of iRFP-PHOSBP in WT and SEC14L2 KO COS-7 cells with OSBP-EGFP or VAPA-EGFP overexpression. Statistics are shown in Fig. 6d. h-k, GI-SIM imaging of endosome fission in NC siRNA transfected WT COS-7 cells (h), VAPs siRNA transfected WT (i), and SEC14L2 KO (j) COS-7 cells and OSBP siRNA transfected SEC14L2 KO cells (k) at 1-s intervals. mEmerald-RAB5 (green) and mCherry-KDEL (magenta) were co-expressed to mark endosomes and ER, respectively. The main panel shows a partial cell periphery with endosome fission. The insets illustrate a single fission event. White arrows mark approximate sites of fission. Time 0 corresponds to the frame in which tubules first emerge. Statistics are shown in Fig. 6e.
